Project Summary Nature uses highly reactive radicals to carry out a diverse set of biochemical functions, many of which are essen- tial to maintaining proper human health. These potent biological radical reactions need to be carried out safely, producing essential specific products, without dangerous side reactions occurring. A large number of such rad- ical reactions are performed by the family of radical SAM enzymes, which use a [4Fe-4S] center with a bound S-adenosylmethionine (SAM) molecule to generate a strongly oxidizing 5'-deoxyadenosyl radical which can in turn drive a large number of difficult chemical reactions. We will target mechanistic aspects of several classes of radical SAM enzymes. Biotin synthase is a radical SAM enzyme that catalyzes the final step in the biosynthesis of the vitamin biotin. A set of Fe-S and radical SAM maturase enzymes are used to build the unique Fe-S center of Fe-Fe hydrogenase, an enzyme which catalyzes the important reduction of protons to dihydrogen and vice versa. And radical SAM enzymes are used to modify many bases in transfer RNA, improving codon-anticodon recognition in order to make protein synthesis more reliable. We are specifically interested in a radical SAM enzyme QueE that is essential for generating 7-deazapurines. This proposal describes a magnetic resonance spectroscopic approach to study such diverse radical SAM enzymes. Specifically, we are using electron para- magnetic resonance (EPR) spectroscopy, which can precisely measure the magnetic environment of unpaired electrons in the radical SAM Fe-S clusters, in the organic radicals that these clusters generate, and in secondary metal centers that are involved in the reactions in many of these enzymes.